Method for detecting down sydrown by non-invasive maternal blood screening

ABSTRACT

The present invention relates to a method for detecting fetal Down syndrome (Trisomy 21), trisomy 13, trisomy 18 and other chromosomal anomalies during prenatal screening by analyzing blood samples from a pregnant woman. More particularly the present invention relates to a method for improving detection efficiency in screening for the anomalies by measuring the amount of the free beta human chorionic gonadotropin (HCG) and nicked or fragmented or aberrant forms of free beta (HCG), all of which are referenced throughout this application as free beta (HCG) in blood samples from pregnant women.

This application is a continuation-in-part of application Ser. No.07/868,160, filed Apr. 14, 1992, which is a continuation-in-part ofapplication Ser. No. 07/420,775, filed Oct. 12, 1989, which is acontinuation-in-part of application 07/360,603, filed Jun. 1, 1989, nowabandoned, which is a continuation-in-part of application Ser. No.07/349,373, filed May 8, 1989, now abandoned, which is acontinuation-in-part of application Ser. No. 07/311,808 filed Feb. 17,1989, now abandoned, which is a continuation-in-part of application Ser.No. 07/297,481, filed Jan. 17, 1989, now abandoned,

FIELD OF THE INVENTION

The present invention relates to a method for detecting fetal Downsyndrome (Trisomy 21), trisomy 13, trisomy 18, Turners syndrome andother chromosomal anomalies during prenatal screening. More particularlythe present invention relates to a method for improving detectionefficiency in prenatal screening for Down syndrome by measuring theamount of free beta (human chorionic gonadotropin "HCG") and nicked orfragmented or aberrant forms of free beta (HCG) all of which arereferenced throughout this application as free beta (HCG).

BACKGROUND OF THE INVENTION

Down syndrome, also referred to as Trisomy 21, is the most commoncongenital cause of severe mental retardation. Generally, fetal Downsyndrome can be determined by diagnostic procedures includingamniocentesis or chorionic villus sampling and karyotyping. However,these diagnostic procedures are invasive and involve risk to the womanand the fetus. For this and other reasons, amniocentesis or chorionicvillus sampling and karyotyping are not routinely performed during allpregnancies. Instead, one or more screening methods may be utilized todetermine when the risk to the pregnancy warrants the risk of undergoingan invasive diagnostic procedure.

The incidence of Down syndrome increases significantly with increasingmaternal age. Historically, the prenatal detection of Down syndrome hasfocused on pregnant women at and over the age of 35, at which ages therisks of Down syndrome approach or exceed the risks of diagnosticprocedures utilized to detect fetal Down syndrome. Therefore thestandard method of prenatal screening has involved selecting women fordiagnostic amniocentesis on the basis of maternal age. Age, however, isan inadequate screening criterion in that only about 20% of all Downsyndrome pregnancies can be detected by carrying out amniocentesis andkaryotyping on the 5% of pregnant women most at risk, that is, thoseaged 35 years or greater. And, because in actual clinical practice onlyabout half of the women aged 35 years or greater undergo amniocentesisand karyotyping, fewer than 10% of Down syndrome pregnancies areprenatally detected.

In 1984 an association between lowered maternal blood alphafetoprotein(AFP) levels and fetal Down syndrome was discovered. For example, see"An association between low maternal serum alpha-fetoprotein and fetalchromosomal abnormalities"; Merkatz, Macri, et al.; Am. J. Obstet.Gynecol. 148:996, 1984; the disclosure of which is hereby incorporatedby reference. In this publication it was noted that other chromosomaltrisomies, in particular Trisomy 13 and Trisomy 18, were also associatedwith lowered maternal blood AFP levels. The incidence of theseadditional chromosomal trisomies (1 in 5000 pregnancies and 1 in 6600pregnancies, respectively) is significantly lower than the general apriori risk associated with Trisomy 21 (Down syndrome, 1 in 800pregnancies). However, because of the association of these otherchromosomal trisomies with lowered MSAFP levels, and elevated ordepressed free beta (HCG) levels, such abnormalities will also bedetected within a screening protocol utilizing maternal blood AFP andfree beta (HCG) and possibly additional markers described herein. It isobvious to those skilled in the art that in using the protocol describedherein for Trisomy 21, the detection of Trisomy 13, Trisomy 18, Turnerssyndrome and other chromosomal anomalies may also be accomplished.

The association between lowered maternal blood AFP levels and fetal Downsyndrome presented the opportunity to use a non-invasive blood screeningtest in the detection of Down syndrome cases in young, apparentlyunaffected families where approximately 80% of Down syndrome casesoccur. It is estimated that the use of a screening test based on lowmaternal blood AFP (as a screening marker) would lead to the prenataldetection of approximately 20% of all cases of fetal Down syndrome.

Another method for screening-involves measuring the level ofunconjugated estriol (UE) in maternal blood. For example, see "Maternalblood screening for Dwon syndrome in early pregnancy"; Wald, et al.British Journal of Obstetrics and Gynocology (BMJ) Volume 95, April1988, the disclosure of which is hereby incorporated by reference.

More recently an association between elevated maternal blood levels ofthe Intact HCG molecule and the alpha subunit of HCG (HCG is composed oftwo subunits) and fetal Down syndrome was discovered. For example, see"Abnormal Maternal Serum Chorionic Gonadotropin Levels in Pregnancieswith Fetal Chromosome Abnormalities"; Bogart, Pandian and Jones;Prenatal Diagnosis, Vol. 7, 623-630 (1987) the disclosure of which ishereby incorporated by reference. In the Bogart article it is estimatedthat the use of elevated maternal blood Intact HCG levels and elevatedmaternal blood levels of the alpha subunit of HCG, would detectapproximately 68% of the chromosomally abnormal fetuses. However, theseresults were obtained from a study on pregnancies at 18-25 weeks ofgestation and the affected cases appear to be of women previouslyidentified as being at risk for Down syndrome.

U.S. Pat. No. 4,874,693 to Bogart discloses an association betweenelevated maternal blood HCG levels and elevated maternal blood levels ofthe alpha subunit of HCG, in weeks 18-25 of pregnancy and fetal Downsyndrome. In the Bogart patent it is estimated that the use of elevatedmaternal blood HCG levels and elevated maternal blood levels of thealpha subunit of HCG in a screening protocol, would detect a greaterpercentage of chromosomally abnormal fetuses than the use of AFP or UEalone. In a paper entitled "Human Chorionic Gonadotropin Levels inPregnancies with Aneuploid Fetuses" (Bogart et al., Prenatal Diagnosis,Vol. 9, 379-384 (1989)), Bogart discloses that a screening methodutilizing HCG and the alpha subunit of HCG is not useful at 9-11 weeksgestation (the first trimester of pregnancy) for selecting pregnanciesat risk for fetal aneuploidy (including Down syndrome).

Generally, as suggested above, screening by evaluation of maternal bloodHCG has involved only the measurement of the Intact HCG molecule andadditionally the measurement of the alpha subunit of HCG. Although thesescreening methods do detect fetal Down syndrome, there is a need and adesire for a method which detects a greater percentage of fetal Downsyndrome cases.

I have discovered a previously unknown association between elevatedlevels of maternal blood free beta (HCG) and fetal Down syndrome. I havealso discovered a previously unknown association between the maternalblood level of free beta (HCG) and the maternal blood level of AFP andfetal Down syndrome. I have further discovered a previously unknownassociation between the ratio of the maternal blood level of free beta(HCG) to the maternal blood level of the intact HCG molecule and fetalDown syndrome. I have still further discovered that using a multivariatediscriminant analysis technique improves the detection efficiency of ascreening method using the maternal blood level of free beta (HCG), orthe maternal blood level of free beta (HCG) and the maternal blood levelof AFP, or the log of either, or the log of both, especially whengestational age is also incorporated as a variable in the discriminantanalysis technique, for a chosen risk cut-off level. Gestational agerefers to the age of the pregnant woman's fetus. Detection efficiencyrefers to the percentage of cases of fetal Down syndrome which arecorrectly detected for a chosen risk cut off level. The risk cut offlevel will be more fully explained in a following section. Discriminantanalysis is a generally known approach to multivariate analysisinvolving the separation of a population into two or more groups on thebasis of probability. Discriminant analysis is also sometimes describedas a way of constructing a linear combination of independent variables,thus reducing the problem of measuring group differences to a univariateproblem. A general discussion of discriminant analysis can be found inMarketing Research; Churchill, G.A.; Dryden, 1976; Chapter 15, pages530-543, the disclosure of which is hereby incorporated by reference. Ihave discovered that subjecting the maternal blood levels of free beta(HCG), the maternal blood levels of intact HCG, the ratio of thematernal blood level of free beta (HCG) to the maternal blood level ofthe intact HCG molecule, the maternal blood level of AFP, the maternalblood level of UE, and gestational age to multi-variate discriminantanalysis detects a greater percentage, with a lower false positive rate,of fetal Down syndrome cases than any other known screening method forthe prenatal detection of Down syndrome. I have further discovered thata still greater number the cases of fetal Down syndrome may be detectedby using only the measurements of the maternal blood levels of free beta(HCG) and the maternal blood levels of AFP and subjecting the log ofeach measurement and gestational age to a multivariate discriminantanalysis. These and other discoveries will be more fully explained inthe Summary of the Invention section and the Detailed Description of theInvention section.

One object of the present invention is to provide a method and processfor screening for fetal Down syndrome which detects a greater percentageof fetal Down syndrome cases with a given false positive rate than otherknown prenatal screening methods.

Another object of the present invention is to provide a method andprocess for screening for fetal Down syndrome which has a lesser falsepositive rate for a given detection percentage than other known methods.

A still further object of the present invention is to applymulti-variate discriminant analysis to methods for screening for Downsyndrome to detect a greater percentage of fetal Down syndrome caseswith a lesser false positive rate.

A further object of the present invention is to provide a method andprocess for screening for fetal Down syndrome by measuring the level ofmaternal blood free beta (HCG).

A still further object of the present invention is to provide a methodand process for screening for fetal Down syndrome by measuring thematernal blood level of AFP and the maternal blood level of free beta(HCG).

Other objects and advantages of the present invention will becomeapparent in the following description of the invention.

SUMMARY OF THE INVENTION

To achieve these and other objects, according to the present invention apregnant woman's (hereinafter sometimes referred to as the patient)maternal blood levels of free beta (HCG) are measured by conventionalimmunological techniques which can include immunoassay techniques suchas those referred to in the papers above, and other techniques known inthe art. The level of free beta (HCG) is then compared to a set ofreference data to determine the patient's risk of carrying a fetus withDown syndrome. To improve detection efficiency, the level of free beta(HCG) and the gestational age can be compared to a set of referencedata. To further improve detection efficiency, a patient's maternalblood levels of free beta (HCG) and AFP (referred to as "markers") aremeasured by conventional immunological methods, including assaytechniques known to the art such as those referred to in the papersabove. The levels of each marker are then compared to a set of referencedata to determine the patient's risk of carrying a fetus with Downsyndrome. A multivariate discriminant analysis technique isadvantageously used to compare the levels of the markers to a set ofreference data. More particularly, a patient specific risk is thencalculated using Bayes rule, the patient's a priori risk, and therelative frequencies for unaffected and affected pregnancies which aredetermined by incorporating the log of the patient's quantitative levelsof each marker into the probability density functions for the referencedata developed using multivariate discriminant analysis. If thepatient's risk of carrying a fetus with Down syndrome is greater than agiven risk cut-off level, the patient should be counseled about furtherdiagnostic tests to confirm the presence of Down syndrome.

Similarly, if the method of the present invention is utilized in ascreening protocol for trisomy 13, trisomy 18, Turner's syndrome orother chromosomal anomalies, the patient's risk of carrying a fetus withthe anomaly may be determined using a multivariate discriminant analysistechnique whereby the level of free beta (HCG) and AFP are compared to aset of reference data.

Incorporating gestational age as a marker along with the level of freebeta (HCG) and the level of AFP will further improve detectionefficiency. Since the maternal blood level of free beta (HCG) and thematernal blood level of AFP for a number of samples tend to bedistributed according to a log-gaussian distribution curve, the greatestdetection efficiency can be achieved by incorporating the log of thepatient's quantitative levels of each marker and gestational age intothe probability density functions for the reference data developed usingmultivariate discriminant analysis.

As described herein, and in my prior applications, free beta (HCG) canexist in many forms including intact free beta (HCG) and so calledaberrant forms of free beta (HCG). These aberrant forms of free beta(HCG) may include fragments of the complete free beta (HCG) molecule and"nicked" free beta (HCG). Additional details concerning these forms, andothers, of free beta (HCG) is contained in the following sections.Immunological methods that measure any or all of the forms of free beta(HCG) fall within the scope of the present invention.

The conventional immunological techniques suitable for use in thepresent invention also include the use of biosensors. A biosensorgenerally comprises a biologically-derived sensing element, such as anantibody, linked to a transducer that can produce an electrical signalor other observable readout. When a certain amount of the substance tobe measured, for example free beta (HCG) or a form thereof, contacts thesensing element a reaction takes place and the transducer translates thereaction into a change of color, fluorecence, temperature, electricalcurrent or other electro-chemical signal. A biosensor that measures thelevel of free beta (HCG) and or a form of free beta (HCG) is within thescope of the method of the present invention.

An advantage of the method and process of the present invention is thatit correctly predicts a higher percentage of fetal Down syndrome cases,with a lesser false positive rate than other known methods andprocesses.

Other advantages of the present invention will become clear from thefollowing more detailed description and the following examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Table, referred to in Example 2, showing the level ofsignificance of individual markers for Trisomy 21.

FIG. 2 is a table, referred to in Example 2, showing Down syndromescreening efficiency of individual markers.

FIG. 3 is a table, referred to in Example 2, showing Down syndromescreening efficiency of composite markers.

FIG. 4 is a table, referred to in Example 2, showing proportion of Downsyndrome cases above given percentiles of the distribution of free beta(HCG) in unaffected pregnancies

FIG. 5 is a table, referred to in Example 2, showing Down syndromeefficiency of individual markers.

FIG. 6 is a table, referred to in Example 2, showing Down syndromescreening efficiency of log AFP and log free beta (HCG) as a compositemarker at different gestational age ranges.

FIG. 7 is a table, referred to in Example 2, showing projected Downsyndrome screening efficiency of AFP, free beta (HCG) and maternal ageacross the U.S.A.

FIG. 8, referred to in Example 2, shows the levels of free beta (HCG) incases of trisomy 21 in relation to various percentlies of the unaffectedpregnancies.

FIG. 9, referred to in Example 2, shows the distributions of free beta(HCG) levels.

FIG. 10 is a Table, referred to in Example 2, showing Down syndromescreening efficiency for a variety of combinations of markers.

FIG. 11 shows the apparatus of the present invention utilized inperforming the method for detecting Down syndrome.

FIGS. 12A; 12B, 13A and 13B show a flowchart for a computer programutilizing the reference parameters for use in conjunction with thedetermination of a patient's specific risk of carrying an affectedfetus.

FIG. 14 shows a flowchart for a computer program utilizing the referenceparameters calculated in the program shown in FIGS. 12 and 13 fordetermining the patient's specific risk of carrying an affected fetus.

FIG. 15, referred to in Example 4, shows the distribution of free beta(HCG) levels in patient samples.

FIG. 16, referred to in Example 5, shows the distribution of free beta(HCG) levels in patient samples.

FIG. 17, referred to in Example 6, shows the distribution of free beta(HCG) levels in patient samples.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention, a maternal blood sample istaken from a patient. The maternal blood level of free beta (HCG) isthen measured by conventional analytical methods, such as immunologicalmethods known to the art. The maternal blood level of free beta (HCG) isthen compared to a set of reference data to determine whether thepatient is at an increased risk of carrying a fetus with Down syndrome.To increase detection efficiency, gestational age and the maternal bloodlevel of free beta (HCG) may be compared to a set of reference data todetermine whether the patient is at increased risk of carrying a fetuswith Down syndrome.

As described in my copending application, filed on the same day as thisapplication and entitled DOWN SYNDROME SCREENING METHOD UTILIZING DRIEDBLOOD SAMPLES, the blood sample taken from the pregnant woman may bedried for transport and future analysis. Thus the method of the presentinvention includes the analysis of both liquid and dried blood samples.

Additionally, it is generally known that certain fragments of free beta(HCG) may be excreted in a pregnant woman's urine. One such fragment iscommonly referred to as the "beta core fragment" and comprises the6th-40th amino acid residues disulfide linked to the 55th-92nd aminoacid residues that make up the complete free beta (HCG) molecule. It ispossible that an assay designed to measure the beta core fragment offree beta (HCG) in a pregnant woman's urine could be utilized in ascreening protocol for Down syndrome according to the method of thepresent invention.

Although any of the known analytical methods for measuring the maternalblood level of free beta (HCG) will function in the present invention,as obvious to one skilled in the art, the analytical method used forfree beta (HCG) must be the same method used to generate the referencedata for free beta HCG. If a new analytical method is used for free beta(HCG), a new set of reference data, based on data developed with themethod, must be generated. Thus, the technique utilized to analyze theblood spots should be the same for the reference data and the samples tobe screened.

It is also generally understood that in generating antibodies specificfor free beta (HCG), some antibodies will be specific for the proteinand some will be specific for carbohydrate associated antigenic sites.The measurement of the level of free beta (HCG) referred to throughoutthe description of the invention includes using antibodies specific foreither the protein or the carbohydrate associated antigenic sites or anyother site on free beta (HCG).

It is further understood by those of ordinary skill in the art, thatwhile the alpha subunit of (HCG) is encoded by a single gene, free beta(HCG) is encoded by a complex family of at least seven very similargenes or pseudogenes. For example, see "Human chorionic gonadotropinbeta-subunit is encoded by at least eight genes arranged in tandem andinverted pairs," Boorstein, Vamvakopoules, & Fiddes; Nature Vol 300, 2Dec. 1982; the teaching of which is hereby incorporated by reference. Itis known that only three of the seven free beta (HCG) genes areexpressed in the normal placental production of free beta (HCG). Forexample, see "Fragmentation of the Beta Subunit of Human ChorionicGonadotropin Produced by Choriocarcinoma; Nishimura, Ide, Utsunomiya,Kitajima, Yuki, and Mochizuki; Endocrinology, Vol. 123, No. 1, 1988; theteachings of which are hereby incorporated by reference. Whether thesesame three genes are expressed in disease states such as during thepresence of fetal Down syndrome, has not been determined. It is,therefore, possible that multiple forms of free beta (HCG) with smalldifferences in amino acid sequences, or other small differences, may besynthesized. It is further possible that in Down syndrome, one or moreof the free beta (HCG) genes are expressed, thereby producing a uniquevariant or variants (previously referenced as nicked or fragmented oraberrant forms) of free beta (HCG). According to the present inventionthese variants could be measured by conventional immunologicaltechniques for measuring free beta (HCG). An assay produced to measurethe specific free beta (HCG) variant, or variants, associated with Downsyndrome may result in even further enhancement of detection efficiency.

We have effectively used assay techniques to measure free beta (HCG) todistinguish between Trisomy 21 affected and unaffected pregnancies.Detection efficiency for Trisomy 21 as high as 83% has been achieved. Asis well known to those skilled in the art, the use of antibodies toquantitate specific analytes may result in degrees of cross-reactivitywith a distinct yet similar substance. Hence, the distinction betweenaffected and unaffected cases may be influenced by the presence of anaberrant form of free beta (HCG) which, because of some degree ofcross-reactivity with the antibodies being used, is being detected. Anaberrant form of free beta (HCG) may be designated as a new biochemicalsubstance. Indeed, information from the scientific literature indicatesthat aberrant forms of free beta (HCG) have been recognized (forexample, see Nishimura et al. infra.)

Trisomy 21 affected cases may also be characterized by an aberrant formof free beta (HCG) in which case those skilled in the art will becapable of developing specific antibodies to such aberrant forms whichmay result in a further enhancement of detection efficiency for thissyndrome.

Alternatively, Down syndrome affected cases may also be characterized bya fragmented form (or fragment) of free beta (HCG) comprising anincomplete portion of the amino acids that comprise free beta (HCG). Aswill be understood by those of ordinary skill in the art, assaysutilized to measure free beta (HCG) will also detect fragments of freebeta (HCG) if the epitope, or epitopes, utilized in the assays arepresent in the fragment of free beta (HCG).

One such aberrant or fragmented form of free beta (HCG) may be referredto as "nicked" free beta (HCG). In nicked free beta (HCG) peptidelinkages are missing between amino acids in free beta (HCG). In knownforms of nicked free beta (HCG) peptide linkages can be missing betweenresidues 44 and 45 or between residues 47 and 48. Techniques have beendeveloped to measure forms of nicked free beta (HCG). As explainedabove, these techniques may be utilized in the method of the presentinvention to screen for fetal Down syndrome by measuring the maternalblood level of nicked free beta (HCG) or the combined blood level ofnicked free beta (HCG) and free beta (HCG).

As illustrated in Example 7, certain immunoassays utilized in themeasurement of free beta (HCG) measure "nicked" free beta (HCG). Themethod of the present invention includes the use of such immunoassays.As illustrated in the Examples, a screening protocol for Down syndromeutilizing immunoassays that measure the level of free beta (HCG) andnicked free beta (HCG) in a pregnant woman's blood, may have a detectionefficiency of approximately 80%.

The reference data reflects the maternal blood level of free beta (HCG)for pregnant women carrying fetuses with Down syndrome (also referred toas affected) and/or the maternal blood level of free beta (HCG) forpregnant women carrying normal fetuses (also referred to as unaffected).As will be generally understood by those of skill in the art, methodsfor screening for fetal Down syndrome are processes of decision makingby comparison. For any decision making process, reference values basedon patients having the disease or condition of interest and/or patientsnot having the disease or condition of interest are needed. In thepresent invention the reference values are the maternal blood level ofthe measured marker or markers, for example, free beta (HCG), in bothpregnant women carrying Down syndrome fetuses and pregnant womencarrying normal fetuses. A set of reference data is established bycollecting the reference values for a number of samples. As will beobvious to those of skill in the art, the set of reference data willimprove by including increasing numbers of reference values.

To determine whether the patient is at increased risk of carrying afetus with Down syndrome, a cut-off must be established. It is obviousto those skilled in the art that a cut-off established to determinewhether a patient is at increased risk of carrying a fetus with Trisomy13 or Trisomy 18 may also be effective in identifying cases of trisomy21. This cut-off may be established by the laboratory, the physician oron a case by case basis by each patient. The cut-off level can be basedon several criteria including the number of women who would go on forfurther invasive diagnostic testing, the average risk of carrying a Downsyndrome fetus to all the women who go on for further invasivediagnostic testing, a decision that any woman whose patient specificrisk is greater than a certain risk level such as 1 in 400 should go onfor further invasive diagnostic testing or other criteria known to thoseskilled in the art. The cut-off level could be established using anumber of methods, including: percentiles, mean plus or minus standarddeviation(s); multiples of median value; patient specific risk or othermethods known to those who are skilled in the art.

In another embodiment of the present invention, which results in adetection of a greater number of the cases of fetal Down syndrome, theblood spots are analyzed for both free beta (HCG) and AFP utilizing adual analyte assay. Although any of the known analytical methods formeasuring the maternal blood levels of these analytes will function inthe present invention, as obvious to one skilled in the art, theanalytical method used for each marker must be the same method used togenerate the reference data for the particular marker. If a newanalytical method is used for a particular marker, a new set ofreference data, based on data developed with the method, must begenerated.

Preferably, a patient specific risk of carrying a fetus with Downsyndrome is calculated using Bayes rule, the patients a priori risk, andthe relative frequencies for unaffected and affected pregnancies whichare determined by incorporating the patient's quantitative levels oneach analyte (free beta (HCG) and AFP) along with the patient'sgestational age, into the probability density functions developed forthe reference data using multivariate discriminant analysis. Themultivariate discriminant analysis can be performed on the commerciallyavailable computer program statistical package Statistical Analysissystem (manufactured and sold by SAS Institute Inc. ) or by othermethods of multivariate statistical analysis or other statisticalsoftware packages known to those skilled in the art.

The probability density function provides a method for comparing thepatient's level of each analyte to a set of reference data. One type ofprobability density function is set forth below, although as will beobvious to one skilled in the art, other probability density functionswill perform similarly, and therefore perform adequately in the presentinvention. ##EQU1## The subscript "a" refers to the affected cases Thesubscript "u" refers to the unaffected cases

(X-M) is a Vector where each element is the level of each variable minusthe mean of the variable.

cov⁻¹ is the inverse of the pooled covariance matrix of the affected andunaffected of all of the variables in the model

(X-M)^(T) is the transpose of the (X-M) vector.

EXP refers to the exponential function.

|COV" refers to the determinant of the covariance matrix of all thevariables in the model for the reference data.

As obvious to those skilled in the art, individual covariance matricesfor unaffected and affected pregnancies can be substituted for thepooled covariance matrix. The formula for the Risk of Down syndromewould then become: ##EQU2##

For the purposes of the discriminant analysis an assumption is made asto the prior probability of Down syndrome in the general unselectedpopulation. Generally, the prior probability is approximately 1 in 800.For the multivariate discriminant analysis a decision is made as to whatrisk cut- off level constitutes a positive test result. For example, ifit is desirable to perform further diagnostic tests on a pregnant womanwho has a 1 in 400 or greater possibility of carrying a Down syndromefetus, then when the results of the discriminant analysis indicate thata pregnant woman has a 1 in 400 or greater possibility of carrying aDown syndrome fetus, the pregnant woman is considered to have a positivetest result. If a positive test result is indicated, the patient shouldbe counseled about further diagnostic tests to confirm the presence ofDown syndrome.

With reference to FIGS. 11-14, the apparatus and a flowchart for acomputer program for calculating the reference parameters and specificrisk are shown.

As shown in FIG. 11, the gestational age GA, the level of AFP and thelevel of free beta (HCG) are determined by conventional techniques fromaffected and unaffected pregnancies in order to develop reference data.A large numb of samples are chosen to increase reliability. Themeasurements for the development of reference parameters are indicatedschematically at 10.

Once the reference parameters 22 are calculated by the processing unit20 after entry via a suitable input device 15, the specific risk 25 fora particular patient can be calculated based on the individual'sspecific measured marker values, indicated at 30.

The program for determining the reference parameters is shown in FIGS.12A and 12B and 13A and 13B and the program for calculating specificrisk is shown in FIG. 14.

With reference now to FIGS. 12A and 12B and 13A and 13B, in a first loop100, the program reads in identification data ID, gestational age GA,quantities of AFP and free beta (HCG) and a CODE indicating whether thepregnancy is affected or unaffected by Trisomy 21 from a reference groupin order to develop reference data. This is shown in step 102. In theflowchart, gestational age GA is denoted by variable X¹, the log of AFPis given by variable X² and the log of free beta (HCG) is given by X³,as shown in step 104. The sum and sum-product matrices are thendetermined or calculated as shown in step 106 based upon the quantitiesX¹, X² and X³. The variable N_(CODE) which count the number of affectedand unaffected cases in the reference group is then incremented. Oncethe loop is terminated, as shown by flow line 110, the means are thencalculated through series of loops defined by the quantities I, J and Kas indicated by reference numeral 112. In these loops, the covariancematrix is calculated utilizing the sum matrix defined in loop 100 andthe sum product matrix calculated in loop 100. After these loops, achoice is made whether to pool or not to pool the covariance matricesfor the affected and unaffected. This choice is inputted in steps 114,116 and 118 the choice is to pool, the covariances are pooled to formpooled covariance matrix as given by step 120, the pooled covariancematrix is inverted resulting in the inverted pooled covariance matrixIPCM as shown at 122, and the means and the inverted pooled covariancematrix are saved in a file and printed out at steps 124 and 126. If thechoice is not to pool the covariance matrices, then each of the twocovariance matrices are inverted in steps 123 and 125 and the means andthe inverted covariance matrices are saved in a file and printed out insteps 125 and 127. These quantities comprise the reference parametersfor the calculation of a specific individual's risk of carrying anaffected fetus.

With reference to FIG. 14, the reference parameters determined duringthe execution of the program shown in FIGS. 12 and 13, the referenceparameters comprising the means and the inverted pooled covariancematrix, are read in as shown at 130. The specific patient record,including the patient identification, the gestational age GA, AFP andfree beta (HCG) are then read in as shown at 132. The gestational age isthen calculated more specifically at 134, and a maternal age calculationis made at 136. At 138, the prior risk is determined based upon maternalage and incidence data. In the examples discussed below, the result ofthis calculation is the factor 1/800, a typical number.

At 140, the prior risk times the relative frequency of carrying anaffected fetus (ABT) is determined, which is the numerator of theequations (1) or (2) discussed above. At 142 the relative frequency ofcarrying an unaffected fetus multiplied by (1-prior risk), (NT), isdetermined, which is the second factor in the denominator of equations(1) or (2) found above. At 144, the specific risk using Bayes Rule isdetermined, i.e., ABN=ABT/(ABT+NT). (Equations (1) and (2)) At 146, theresults are printed, i.e., the patient's specific risk ABN and thepatient identification number.

As will be apparent to a person of skill in the art, other statisticaland mathematical techniques for calculating the reference parameters,other than a linear discriminant analysis procedure, can also be used.

According to a preferred embodiment of the present invention a maternalblood sample is taken from a patient. The maternal blood levels of freebeta (HCG) and AFP (hereinafter referred to as "markers") are thenmeasured by conventional immunological methods known to the art.Although any of the known analytical methods for measuring the maternalblood levels of these markers will function in the present invention, asobvious to one skilled in the art, the analytical method used for eachmarker must be the same method used to generate the reference data forthe particular marker. If a new analytical method is used for aparticular marker, a new set of reference data, based on data developedwith the method, must be generated.

A patient specific risk of carrying a fetus with Down syndrome is thencalculated using Bayes rule, the patient's a priori risk, and therelative frequencies for unaffected and affected pregnancies which aredetermined by incorporating the patient's quantitative levels of eachmarker along with the patient's gestational age, into the probabilitydensity functions developed for the reference data using multivariatediscriminant analysis. To further increase detection efficiency, the logof the patient's quantitive levels of free beta (HCG) and AFP, alongwith the patients gestational age, are incorporated into the probabilitydensity functions developed for the reference data using multivariatediscriminant analysis. The multivariate discriminant analysis can beperformed on the commercially available computer program statisticalpackage Statistical Analysis System (manufactured and sold by SASInstitute Inc. ) or by other methods of multivariate statisticalanalysis or other statistical software packages known to those skilledin the art.

For the purposes of the discriminant analysis an assumption is made asto the prior probability of Down syndrome in the general unselectedpopulation. Generally, the prior probability is approximately 1 in 800.For the multivariate discriminant analysis a decision is made as to whatrisk cutoff level constitutes a positive test result. For example, if itis desirable to perform further diagnostic tests on a pregnant woman whohas a 1 in 400 or greater possibility of carrying a Down syndrome fetus,then when the results of the discriminant analysis indicate that apregnant woman has a 1 in 400 or greater possibility of carrying a Downsyndrome fetus, the pregnant woman is considered to have a positive testresult. If a positive test result is indicated, the patient should becounseled about further diagnostic tests to confirm the presence of Downsyndrome.

As obvious to one skilled in the art, in any of the embodimentsdiscussed above, changing the risk cut-off level of a positive or usingdifferent a priori risks which may apply to different subgroups in thepopulation, could change the results of the discriminant analysis foreach patient.

The present invention is not limited to the embodiments discussed abovebut rather includes all of the possible embodiments and combination ofmarkers disclosed in the following examples.

EXAMPLE 1

Over 400 patient samples were utilized to study the relationship offetal Down syndrome to the maternal blood levels of free beta (HCG) inconjunction with maternal serum AFP (MSAFP) , UE, and intact HCG. Thesesamples included 25 maternal blood samples from pregnant women known tobe carrying fetuses with Down syndrome and control samples matched tothe affected cases. In this example liquid blood samples were analyzed.

For each blood sample quantitative levels of AFP, the intact HCGmolecule, free beta (HCG), and UE (hereinafter each is referred to as aMarker) were determined by the following assay techniques:

    ______________________________________                                        Marker     Assay Technique                                                    ______________________________________                                        MSAFP      Enzyme linked immunosorbent assay (ELISA)                          UE         Radioimmunoassay                                                   Intact HCG Bead type ELISA                                                    free beta (HCG)                                                                          ELISA                                                              ______________________________________                                    

The level of each Marker became a variable in the stepwise discriminantprocedure and the linear discriminant procedure on the commerciallyavailable computer software statistical package Statistical AnalysisSystem (SAS Institute Inc. ) to generate a set of reference data. Theratio of free beta (HCG) to the intact HCG molecule and the patientsgestational age were also incorporated as variables. The stepwisediscriminant procedure determined that all of the variables could beincorporated into the linear discriminant procedure. The lineardiscriminant procedure was then performed on each variable separatelyand on different combinations of variables. The results of thesediscriminant analyses are summarized in the chart below. Sensitivity isthe percentage of fetal Down syndrome cases which show a positive testresult. False positives is the percentage of normal fetuses which show apositive test result.

    ______________________________________                                                                      FALSE                                           VARIABLE          SENSITIVITY POSITIVES                                       ______________________________________                                        MSAFP             15.4%       4.2%                                            UE                15.4%       2.8%                                            Intact HCG        37%         8.6%                                            MSAFP, UE Intact HCG                                                                            50.0%       7.2%                                            free beta (HCG) + Intact HCG                                                                    60.0%       8.5%                                            Composite w/o ratio                                                                             76%         5.3%                                            Composite w/o UE  76%         5.3%                                            Composite         80%         4.3%                                            Composite w/o free beta HCG                                                                     60.0%       5.3%                                            *log intact HCG + 68%         7.6%                                            (log free beta (HCG) +                                                        intact HCG)                                                                   *log intact HCG, log MSAFP                                                                      88%         7.4%                                            (log free beta (HCG) +                                                        intact HCG)                                                                   ______________________________________                                         Composite = MSAFP + free beta (HCG) + Intact HCG + UE + Ratio Gestational     age is incorporated along with each variable Risk cutoff level = 1 in 400     except (*) which is 1 in 365.                                            

As obvious to one skilled in the art, changing the risk cut-off level ofa positive, or use of different a priori risks which may apply todifferent subgroups in the population will change the results of thediscriminant procedure for the patient.

EXAMPLE 2

Over 550 patient samples were utilized to study the relationship offetal Down syndrome to the maternal blood levels of free beta HCG.Initially 29 samples from pregnant women known to be carrying fetuseswith Down syndrome and 520 unaffected samples matched for gestationalage (same week), maternal age (within 3 years) and freezer storage time(within one month) were analyzed. All samples were from singleton,non-diabetic, white gravid women. In this Example each of the samplesanalyzed was a liquid blood sample.

In order to avoid training set bias in estimates of screeningefficiency, the concept of a validation set was used. A validation setis a set of data independent of the reference data set. The results frompatients in the validation set are not used in establishing referencedata. Rather, results from patients in the validation set are comparedto the reference data set to determine screening efficiency. This secondvalidation set consisted of 26 additional, confirmed cases of trisomy 21(55 cases total) and a randomly selected group of 159 control samples.The control samples were similarly drawn from singleton, non-diabetic,white gravid women.

The total study consisted of 4388 determinations on 7 different assaysof the maternal blood levels of the markers a set forth below:

    ______________________________________                                        Marker    Assay                                                               ______________________________________                                        MSAFP     ELISA (enzyme linked immunosorbent assay)                           Intact HCG                                                                              ELISA                                                               Intact hcg +                                                                            ELISA                                                               free beta HCG                                                                 free beta HCG                                                                           RIA (radio immunoassay)                                             free alpha-HCG                                                                          RIA                                                                 UE        2 methods, Enzyme Immunoassay & RIA                                 ______________________________________                                    

The level of each Marker became a variable in the stepwise discriminantprocedure and the linear discriminant procedure on the commerciallyavailable computer software statistical package Statistical AnalysisSystem (SAS Institute Inc.) to generate a set of reference data.Gestational age wac also incorporated as a variable. The lineardiscriminant procedure was then performed on each variable separatelyand or different combinations of variables. Patients were classified asbeing affected or unaffected based on a Down syndrome risk cut-off of 1in 365. Unaffected cases that were classified as affected wereconsidered false positive. Each patient's risk of Down syndrome wascalculated using Bayes' rule, the multivariate normal probabilitydensity functions for affected and unaffected cases, and a general apriori risk of 1 in 800. A pooled covariance matrix was used for eachprobability density function.

The results found in Tables 1 through 3, shown in FIGS. 1-3, relate tothe initial study set. Results in tables 5-7, FIGS. 5-7, are based onthe classification of patients in the validation set. Table 4, shown inFIG. 4, and FIGS. 8 and 9 are based on the initial study set and allaffected cases.

The results from the assay procedures were analyzed to determine whetherthere existed significant differences in the levels of each markerbetween affected and unaffected cases. Table 1 (FIG. 1) indicates thataffecteds were significantly different from unaffected in all but U.E..Additionally, the false positive rate and detection efficiency; of eachmarker was determined as shown in Table 2 (FIG. 2). The highestdetection efficiency was achieved with an HCG assay measuring both theintact molecule and free beta (HCG). A further enhancement in detectionefficiency was observed by combining individual markers into composites.The composite containing the HCG assay, which measured both the intactHCG molecule and free beta (HCG), produced the highest detectionefficiency among the composites as shown in Table 3 (FIG. 3).

The evaluation of the beta and alpha subunit of HCG individually showedthat no significant differences existed between affected and unaffectedcases for the alpha subunit (p 0.23) while a significant increase infree beta (HCG) was observed in affecteds (p=0.001). As generally knownin the art, p measures the strength of evidence in scientific studies byindicating the probability that a result at least as extreme as thatobserved would occur by chance. The lower the p value the stronger theevidence that the observation is not a chance occurrence.

FIG. 8 shows the 10th, 50th and 90th percentiles of free beta (HCG) bygestational age. A continuing downward trend by gestational age inunaffected pregnancies is observed. Analysis of free beta (HCG) levelsin cases of fetal Down syndrome, as shown in Table 4 (FIG. 4), revealsthat 86% fall above the median of unaffected.

The levels of free beta (HCG) in both the affected and unaffected casesfit a log Gaussian distribution (p=0.78 and 0.86). FIG. 9 illustratesthese distributions. Table 5 (FIG. 5) provides detection efficiency dataon free beta (HCG) and free alpha (HCG) individually. The high detectionefficiency of free beta (HCG) is shown in Table 5.

An even higher detection efficiency was achieved with a composite of AFPand free beta (HCG). By incorporating the log of the level of free beta(HCG) and the log of the level of MSAF into the linear discriminantprocedure on the commercially available computer software statisticalpackage Statistical Analysis System (SAS Institute Inc.), as describedabove, a superior detection efficiency was achieved as shown in Table 6(FIG. 6). A high detection efficiency would also be achieved using thelevel of free beta (HCG) and the level of AFP, as opposed to the log ofeach.

Further analysis of the data noted that both AFP and free beta (HCG) areindependent of maternal age (p=0.8394 and 0.5214 using Kruskal-Wallistests across four different maternal age groups for AFP and free beta(HCG) respectively, (ages=30, 31-35, 36-40, and 40). Additionally thecorrelation (r) of the levels of free beta (HCG) and AFP was notsignificantly different from zero (r=0.04, p=0.39 and r=-0.06, p=0.81for unaffected and affected cases respectively).

The fundamental observation found in our data confirms .the fact thatfree beta (HCG) contributes the highest detection efficiency for Downsyndrome. In fact, the use of an assay measuring solely free beta (HCG)produced a detection efficiency and false positive rate of 65.4% and5.2% respectively. These rates are comparable to those reported byothers utilizing a combination of three assays. Thus, as previously setforth, reducing the number of assays is an advantage of the presentinvention.

Our findings on the contribution of free beta (HCG) are based on thefollowing: (a) the single best contributor to detection efficiency forDown syndrome was an assay for free beta (HCG), (b) an assay for theintact HCG molecule yields substantially lower detection rates, (c) anassay which measures a combination of the intact HCG molecule and freebeta (HCG) yields a higher detection efficiency than an assay measuringthe intact HCG molecule alone.

It is established that the risk of fetal Down syndrome increases withmaternal age. Therefore, as described above, to produce patient-specificrisks for clinical use of the present invention a maternal age specifica priori risk is incorporated into the multivariate discriminantanalysis procedure. Since both AFP and free beta (HCG) levels areindependent of maternal age, we have analyzed our data to see how manyunaffected and affected women would have positive results given an apriori risk for each individual age. The foregoing information was usedto project, based upon the maternal age distribution of live births inthe United States, the false positive and sensitivity rate forcomprehensive nationwide screening within the United States. As shown inTable 7 (FIG. 7) the projections indicate that it is possible to achievea detection rate of 80%, with 5% false positives.

The samples described in Example 2 were further analyzed to discover thedetection efficiency of other combinations of the markers. Moreparticularly, the linear discriminant procedure of Example 2, with thesame risk cut-off and a priori risk level, was performed on differentcombinations of the markers, multiples of the median (MOM) for themarker and logs of the markers, with and without the incorporation ofgestational age. The linear discriminant procedure was performed usingboth the reference data and the validation data. The results aresummarized in Table 8 in FIG. 10.

EXAMPLE 3

The following example illustrates the preparation of a one step freebeta (HCG) assay and a two step free beta (HCG) assay.

Preparation of a One Step Free Beta (HCG) Assay

1. A ninety-six well microtiter plate is coated with a catching antibodythat is specific to the free beta human chorionic gonadotropin (HCG)molecule. The antibody may be either monoclonal or polyclonal. Theconcentration of antibody used to coat the plate is 0.8 micrograms perwell but could be different, if desired. The plate is incubated at 4° C.for at least 16 hours.

2. The plate is washed with a phosphate buffered saline solution of Ph7.2 that contains 0.05% Tween 20. Other suitable wash buffers may beemployed. The plate is then blocked with a solution containing 3%hydrolyzed animal protein and 0.05% Tween 20 in a phosphate bufferedsaline solution of Ph 7.2. Other solutions, familiar to those skill inthe art, such as a 1% bovine serum albumin solution, may be used. Threehundred microliters of the blocking solution is added to each well andthe plate is allowed to incubate for an hour at ambient temperature.Other blocking procedures are also viable, e.g. glazing.

3. The plate is then washed, as described earlier, and 100 microlitersof assay buffer containing a biotinylated antibody specific to the freebeta (HCG) is added to each well. The assay buffer used is 3% hydrolyzedanimal protein and 0.05% Tween 20 in a phosphate buffered salinesolution of Ph 7.2, but may be any of a number of suitable solutionsknown to those skilled in the art. The antibody may be monoclonal orpolyclonal and, depending on the preference of the operator, may beconjugated to a substance other than biotin, such as horseradishperoxidase or alkaline phosphatase. The concentration of the antibody inthe assay buffer may be adjusted to obtain optimal absorbance values.

4. Twenty microliters of sample is then added to each well. The samplemay be: assay buffer, run as blank to verify the performance of theassay; a solution of free beta (HCG) used to standardize values ofunknown samples; or a serum sample from second trimester gravid-woman.The plate is vortexed for 30 seconds and then placed on a rotator Q 200rpm, where it incubates for 30 minutes at ambient temperature.

5. The plate is then washed as described previously. One hundredmicroliters of assay buffer containing streptavidin conjugated tohorseradish peroxidase is then added to each well. This step is notrequired if the second antibody used i conjugated to a substance otherthan biotin. The concentration of streptavidin-peroxidase in assaybuffer is 2.0 micrograms per milliliter. The plate is placed on arotator Q 200 rpm for 5 minutes at ambient temperature.

6. The plate is then washed as described previously. One hundredmicroliters of an o-phenylendiamine substrate solution is added to eachwell. This substrate solution may alternatively be any one of a numberappropriate dyes known to those skilled in the art and depends upon whatsubstance is conjugated to the second antibody. The plate is placed on arotator Q 200 rpm and incubated at ambient temperature in the dark for 8minutes.

7. One hundred microliters of dilute (1.0 N) sulfuric acid is then addedto each well to stop the reaction of the substrate.

8. The absorbance of each well is determined spectrophotometrically at492 nm.

Preparation of Two-Step Beta (HCG) Assay

1. A ninety-six well microtiter plate is coated with a catching antibodythat is specific to the free beta (HCG) molecule. The antibody may beeither monoclonal or polyclonal. The concentration of antibody used tocoat the plate is 0.8 micrograms per well but may be different, ifdesired. The plate is incubated at 4° C. for at least 16 hours.

2. The plate is washed with a phosphate buffered saline solution of Ph7.2 that contains 0.05% Tween 20. Other suitable wash buffers may beemployed. The plate is then blocked with a solution containing 3%hydrolyzed animal protein and 0.05% Tween 20 in a phosphate bufferedsaline solution of Ph 7.2. Other solutions, familiar to those skilled inthe art, such as a 1% bovine serum albumin solution, may be used. Threehundred microliters of the blocking solution is added to each well andthe plate is allowed to incubate for on hour at ambient temperature.Other blocking procedures, such as glazing, may be employed.

3. The plate is then washed, as described earlier, and 100 microlitersof an assay buffer is added to each well. The assay buffer used is 3%hydrolyzed animal protein and 0.05% Tween 20 in a phosphate bufferedsaline solution of Ph 7.2, but may be any of a number of suitablesolutions known to those skilled in the art.

4. Twenty microliters of sample is then added to each well. The samplemay be: assay buffer, run as the blank to verify the performance of theassay; a solution of free beta (HCG) used to standardize values ofunknown samples; or a serum sample from second trimester gravid woman.The plate is vortexed for 30 seconds and then placed on a rotator 200rpm, where it incubates for 30 minutes at ambient temperature.

5. The plate is then washed, as described previously, and 1o microlitersof assay buffer containing a biotinylated antibody specific to the freebeta (HCG) is added to each well The antibody may be monoclonal orpolyclonal and, depending or the preference of the operator, may beconjugated to a substance other than biotin, such as horseradishperoxidase or alkaline-phosphatase. The concentration of the antibodymay adjusted to obtain optimal absorbance values. The plate is vortexedfor 30 seconds and then placed on a rotator 200 rpm where it incubatesfor 30 minutes at ambient temperature.

6. The plate is then washed as described previously. One hundredmicroliters, of assay buffer containing streptavidin conjugated tohorseradish peroxidase is then added to each well. This step is notrequired if the second antibody used is conjugated to a substance otherthan biotin. The concentration of streptavidin-peroxidase in assaybuffer is 2.0 micrograms per milliliter. The plate is placed on arotator @200 rpm for 5 minutes at ambient temperature.

7. The plate is then washed as described previously. One hundredmicroliters of an o-phenylenediamine solution is added to each well.This substrate solution may alternatively be an one of a numberappropriate dyes known to those skilled in the art and depends upon whatsubstance is conjugated to the second antibody. The plate is placed on arotator @200 rpm and incubated at ambient temperature in the dark for 8minutes.

8. One hundred microliters of dilute (1.0 N) sulfuric acid then added toeach well to stop the reaction of the substrate.

9. The absorbance of each well is determined spectrophotometrically at492 nm.

These two assays were used in carrying out the method of the presentinvention. One hundred seventy eight (178) patient liquid blood sampleswere utilized to study the relationship of fetal Down syndrome to thematernal blood levels of free beta HCG. Twenty six (26) samples frompregnant women known to be carrying fetuses with Down syndrome and 152unknown unaffected samples were analyzed. All samples were fromsingleton, non-diabetic, white gravid women.

The patient samples were then analyzed for quantitative levels of MSAFP,by an ELISA assay, and levels of free beta (HCG) by the one step assayand the two step assay independently. The level of MSAFP and the levelof free beta (HCG) by each assay then became a variable in the lineardiscriminant procedure on the commercially available computer softwarestatistical package Statistical Analysis System to generate a set ofreference data. The patient's gestational age was also incorporated as avariable in the discriminant procedure. The results of thesediscriminant analyses, for all gestational ages, and for gestationalages between 14 and 16 weeks are summarized below.

    ______________________________________                                                 False  Detection                                                              Positive                                                                             Efficiency Controls Affected                                  ______________________________________                                        ALL WEEKS                                                                     Log (beta-1)                                                                             6.6%     69.2%      152    26                                      Log (beta-1) +                                                                           5.9%     72.0%      152    25                                      Log (AFP)                                                                     Log (beta-2)                                                                             8.2%     33.3%      138    18                                      Log (beta-2) +                                                                           10.1%    64.7%      138    17                                      Log (AFP)                                                                     Log (beta-2)*                                                                            9.6%     33.3%      136    18                                      Log (beta-2) +                                                                           10.3%    52.9%      136    17                                      Log (AFP)*                                                                    Weeks 14-16                                                                   Log (beta-1)                                                                             5.8      68.4%      104    19                                      Log (beta-1) +                                                                           4.8%     73.7%      104    19                                      Log (AFP)                                                                     Log(beta-2)                                                                              7.1%     45.4%      98     11                                      Log(beta-2) +                                                                            9.2%     63.6%      98     11                                      Log (AFP)                                                                     Log (beta-2)*                                                                            10.4%    54.6%      96     11                                      Log (beta-2) +                                                                           8.3%     63.6%      96     11                                      Log (AFP)*                                                                    ______________________________________                                         Note: All analyses included gestational age                                   *In the analyses with a * next to them 2 outliers with values of 260 and      316 were removed.                                                        

The suffix -1 means one-step procedure, -2 means two-step procedure.

Detection Efficiency refers to the percentage of fetal Down Syndromecases which show a positive test result.

False Positive refers to the percentage of normal fetuses which show apositive test result.

Controls refers to the number of unaffected samples analyzed.

Affected refers to the number of affected samples analyzed.

Using a combination of the one-step assay for free beta (HCG) and AFPyields the highest detection efficiency with the lowest false positiverate for all weeks of gestation and for 14-16 weeks gestation.

Our findings support the performance of Down syndrome screening in afeasible and effective fashion within a prenatal serum screeningprotocol which includes: (a) non-invasive techniques (b) high detectionefficiency with low false positive rates, (c) use of markers which arelargely independent of each other (d) absence of unwieldy restrictionsin blood sampling (time of day, diet, personal habits, etc.) and (e)compatibility with other prenatal screening services.

EXAMPLE 4

This example illustrates the use of the method of the present inventionin screening for Down syndrome during the first trimester of pregnancy.Maternal serum samples from 150 patients were utilized. Each patient wascarrying a fetus that, at the time the sample was taken, was between 9and 13 weeks of gestational age. 139 of the patients were carrying afetus without Down syndrome, referred to herein as "controls" and 11patients were carrying a fetus with Down syndrome. Each of the patientswas a non-diabetic, white, woman with a singleton pregnancy.

The level of free beta in each of the 150 maternal serum samples wasanalyzed utilizing the two step free beta (hCG) assay described inExample 3. The results of this analysis are shown graphically in FIG.15. As shown in FIG. 15, the median level of free beta in the maternalserum of patients carrying a fetus with Down syndrome was 2.45 MOM(multiples of the median) greater than the median level of free beta inthe maternal serum of the controls. This level of free beta (HCG) inpatients carrying a fetus with Down syndrome is significantly greaterthan the level of free beta (HCG) in the controls. Thus, the free betaprotein is significantly elevated in the first trimester when Downsyndrome is present.

EXAMPLE 5

This example also illustrates the use of the method of the presentinvention in screening for Down syndrome during the first trimester ofpregnancy. Maternal serum samples from 277 patients were utilized. Eachpatient was carrying a fetus that, at the time the sample was taken, wasbetween 7 and 13 weeks of gestational age. 264 of the patients werecarrying a fetus without Down syndrome, referred to herein as "controls"and 13 patients were carrying a fetus with Down syndrome. Each of thepatients was a non-diabetic, white, woman with a singleton pregnancy.

The level of free beta (HCG) in each of the 277 maternal serum sampleswas analyzed utilizing the two step free beta (HCG) hCG assay describedin Example 3. The results of this analysis are shown graphically in FIG.16. As shown in FIG. 16, the median level of free beta (HCG) in thematernal serum of patients carrying a fetus with Down syndrome was 1.82MOM (multiples of the median) greater than the median level of free betain the maternal serum of the controls. This level of free beta (HCG) inpatients carrying a fetus with Down syndrome is significantly greaterthan the level of free beta (HCG) in the controls. Thus, the free betaprotein is significantly elevated in the first trimester when Downsyndrome is present.

EXAMPLE 6

This example further illustrates the use of the method of the presentinvention in screening for Down syndrome during the first trimester ofpregnancy. Maternal serum samples were drawn from 392 patients who hadchosen to undergo a chorionic villus sample (CVS) procedure. The CVSprocedure is a clinical technique wherein a sample of chorionic villi isobtained for chromosomal analysis karotyping. The serum samples utilizedin this example were drawn from the patients prior to the patientsundergoing the CVS procedure. Usually, this technique is utilized withpatients 35 years old, or older, a group that is known to have a higherincidence of carrying a fetus with Down syndrome, or utilized withpatients having a family history of chromosomal abnormalities.

Each patient was carrying a fetus that, at the time the sample wastaken, was between 9 and 13 weeks of gestational age. 386 of thepatients were carrying a fetus without Down syndrome, referred to hereinas "controls" and 6 patients were carrying a fetus with Down syndrome.Each of the patients was a non-diabetic, white, woman with a singletonpregnancy.

The level of free beta (HCG) in each of the 392 maternal serum sampleswas analyzed utilizing the two step free beta (HCG) assay described inExample 3. The results of this analysis are shown graphically in FIG.17. As shown in FIG. 17, the median level of free beta (HCG) in thematernal serum of patients carrying a fetus with Down syndrome was 2.94MOM (multiples of the median) greater than the median level of free beta(HCG) in the maternal serum of the controls. This level of free beta(HCG) in patients carrying a fetus with Down syndrome is significantlygreater than the level of free beta (HCG) in the controls. Thus, thefree beta protein is significantly elevated in the first trimester whenDown syndrome is present.

The Examples further illustrate that the method of the presentinvention, as described herein, may be utilized to non-invasively screenfor Down syndrome during the first or second trimester of pregnancy. Asused herein, the first trimester of pregnancy refers to the time periodprior to 14 weeks of gestational age. Similarly, the second trimester ofpregnancy refers to the time period between 14 and 26 weeks ofgestational age.

For example, a laboratory could generate a set of gestational weekspecific reference data containing the levels of free beta (HCG) inmaternal serum samples obtained during the first trimester of pregnancy.This reference data can be generated in a manner comparable to themanner set forth in the Examples.

To screen for fetal Down syndrome, the laboratory would measure andcompare the level of free beta (HCG) in a blood sample obtained from apatient during the first trimester of pregnancy to the reference data todetermine whether the patient is at sufficient risk of carrying a fetuswith Down syndrome to warrant further testing. As set forth herein, thisdetermination by the laboratory requires a comparison of the patient's aposteriori (after screening) risk of carrying a fetus with Down syndromewith an established cut-off level which reflects the risk to the patientand fetus of an invasive procedure to obtain fetal cells for chromosomalanalysis.

A laboratory could decide to use a multiples of the median approach todetermine a positive screening result. For example, using the data setforth in Example 4 as a set of reference data, the reference data wouldindicate that the median level of free beta (HCG) is 2.45 times greaterin women carrying Down syndrome fetuses than in women carryingunaffected fetuses. Using a multiples of the median approach toscreening, if the level of free beta (HCG) in the patient's sample isgreater than a chosen cut-off level such as 2.5 multiples of the mediangreater than the level of free beta (HCG) in the controls, thelaboratory would recommend that the pregnant woman undergo furtherinvasive testing to diagnose whether her fetus has Down syndrome.

It should be noted that this screening method, relying on a Multiples ofthe Median approach for a single analyte, may not be as accurate as ascreening procedure utilizing multiple analytes and a multivariatediscriminant analysis approach, such as that described in Example 1.

Example 7

This example illustrates that certain immunoassays utilized in themeasurement of free beta (HCG) will also measure "nicked" free beta(HCG).

Nicked free beta (HCG) was prepared as follows. 100 μl of 212 μg/ml hCGβ-subunit (Scripps Catalog Number C0904) manufactured and sold byScripps Laboratories, LaHoya Calif. diluted in deionized water was addedto 100 μl of hLE (0.3 unit) (hLE=human leucocyte elastase) prepared in0.1 M Tris-HCl, ph 8.5 containing 0.5 M NaCl and 1 g Brij per liter,manufactured and sold by Sigma Chemical Company, St Louis, Mo.. Themixture was incubated at 37° C. Samples were drawn at 0, 60, 120 and 180minutes from the mixture. 10 μl of 10 mg/ml soybean trypsin inhibitorwas added to each sample to stop the reaction between the β HCG and thehLE, thereby stopping the formation of further nicked free beta (HCG).The four samples, one of free beta (HCG) (0 minute sample) and threecontaining nicked free beta (HCG) (60, 120 and 180 minute samples), wereanalyzed as follows.

Seven additional concentrations of each sample were made by diluting theinitial sample by half, in a stepwise fashion, using PBS (phosphatebuffer saline solution) pH 7.2 containing stabilizer and preservative.Thus eight concentrations of each sample were prepared, 100%, 50%, 25%,12.5%, 6.25%, 3.125%, 1.5625%, 0.78125% (%=% initial sample). The eightconcentrations of each sample (0, 60, 120 and 180 minutes) were analyzedtwice utilizing the two step free beta (HCG) assay described herein inExample 3, with two different pairs of catching/detecting antibodies.The results were as follows.

In each assay, a slight elevation occurred in measured level of freebeta (HCG) in the samples incubated for 60, 120 and 180 minutes with hLEin comparison with the measured level of free beta (HCG) in the sampleincubated for 0 minutes. The average % change for each assay was 102.1%(range 96.2%-107.6%) and 112.4% (range 106.3% to 121.7%) respectivelyfor 180 minute samples versus the 0 minute samples for the eightconcentrations tested.

In the first antibody pair, recognition of an increasing nicked freebeta (HCG) percentage in the samples did not alter the degree ofimmunoreactivity to free beta (HCG) significantly. The second antibodypair had more affinity for nicked free beta (HCG) than the firstantibody pair. In the case of the second antibody pair, the degree ofchange was increased over the span from time 0 minutes to 60 minutes(110.5% change); and 0 to 120 minutes (115.1% change). The % change at180 minutes did not increase versus the % change at 120 minutes. Theaverage percent change for the second antibody pair was 112.7%. Thus,the first antibody pair is equally reactive to free beta (HCG) andnicked free beta (HCG) and the second antibody pair is more reactive(+12%) to nicked free beta (HCG) than the first antibody pair.

These results provide support for the use of an assay that measures bothnicked free beta (HCG) and free beta (HCG) in a screening protocol forDown Syndrome. The use of such an assay, in the method of the presentinvention, should be expected to provide detection efficiencies ofapproximately 80%.

Our results indicate that it is possible to achieve higher detectionrates in Down syndrome screening while conducting fewer biochemicalanalyses than proposed by others. The use of highly effective markers inDown syndrome screening can serve to provide non-invasive screeninginformation during the early antenatal period to the highest proportionof families affected by this most common cause of severe mentalretardation.

As obvious to one skilled in the art changing the risk cut-off level ofa positive, or using different a priori risks which may apply todifferent subgroups in the population, will change the results of thediscriminant procedure for the patient.

Accordingly, it should be clearly understood that the present inventionincludes all modifications falling within the scope of the followingclaims.

I claim:
 1. A screening method for determining a pregnant woman's riskof carrying a fetus with Down syndrome comprising: measuring saidpregnant woman's maternal blood for free beta (human chorionicgonadotropin (HCG)) level during a time period selected from the groupconsisting of: the first trimester of pregnancy; the second trimester ofpregnancy and the third trimester of pregnancy; and comparing said levelof free beta (HCG) to reference values of the level for free beta (HCG)during the time period in: (1) pregnant women carrying Down syndromefetuses and (2) pregnant women carrying normal fetuses, said comparisonbeing indicative of said pregnant woman's risk of carrying a fetus withDown syndrome, wherein a higher level of free beta (HCG) is indicativeof a higher probability of carrying a fetus with Down syndrome.
 2. Themethod of claim 1 further comprising measuring the pregnant woman'smaternal blood for alpha-fetoprotein (AFP) level during the time periodand incorporating reference values of the levels AFP during the timeperiod of pregnancy in: (1) pregnant women carrying Down syndromefetuses and (2) pregnant women carrying normal fetuses, into saidcomparison wherein a lower level of AFP is indicative of a higherprobability of risk. is carrying a fetus with Down syndrome comprising:measuring
 3. The method of claim 1 wherein the measurement is performedutilizing a biosensor.
 4. A method for determining the risk that apregnant woman is carrying a fetus with Down syndrome comprising:measuring said pregnant woman's maternal blood level of a fragment offree beta (human chorionic gonadotropin (HCG)) during a time periodselected from the group consisting of the first trimester of pregnancy;the second trimester of pregnancy and the third trimester of pregnancyand comparing the measurement of said level of the fragment of free beta(HCG) to reference data containing reference values of the level of thefragment of free beta (HCG) during the time period in: (1) pregnantwomen carrying Down syndrome fetuses and (2) pregnant women carryingnormal fetuses, said comparison being indicative of the pregnant woman'srisk wherein a higher level of the fragment of free beta (HCG) isindicative of a higher probability of carrying a fetus with Downsyndrome.
 5. The method of claim 4 wherein the fragment of free beta(HCG) is selected from the group consisting of: the protein portion offree beta (HCG; the carbohydrate portion of free beta (HCG); and theportion of free beta (HCG) located at about the junction of thecarbohydrate and the protein portions of free beta (HCG).
 6. The methodof claim 5 further comprising measuring the pregnant woman's maternalblood for alpha-fetoprotein (AFP) level during the time period andincorporating reference values of the levels AFP during the time periodin: (1) pregnant women carrying Down syndrome fetuses and (2) pregnantwomen carrying normal fetuses, into said comparison wherein a lowerlevel of AFP is indicative of a higher probability of risk.
 7. A methodfor determining whether a pregnant woman's risk of carrying a fetus withDown syndrome warrants further testing comprising: measuring saidpregnant woman's maternal blood level of an analyte during a time periodselected from the group consisting of the first trimester of pregnancy;the second trimester of pregnancy and the third trimester of pregnancy;using an assay for free beta (HCG) and comparing said level of saidanalyte to a set of reference data containing reference values atvarious gestational ages of the level of the analyte during the timeperiod in pregnant women carrying Down syndrome fetuses and pregnantwomen carrying normal fetuses, said comparison being indicative ofpregnant woman's risk of carrying a fetus with Down syndrome wherein ahigher level of the analyte is indicative of a higher probability ofcarrying a fetus with Down syndrome.
 8. The method of claim 1, 2, or 7wherein the free beta (HCG) is an aberrant form of free beta (HCG). 9.The method of claim 8 wherein the free beta (HCG) is nicked free beta(HCG).
 10. A screening method for determining a pregnant woman's risk ofcarrying a fetus with Down syndrome comprising: measuring said pregnantwoman's urine level of free beta (human chorionic gonadotropin (HCG))level during a time period selected from the group consisting of: thefirst trimester of pregnancy; the second trimester of pregnancy and thethird trimester of pregnancy; and comparing said level of free beta(HCG) to reference value of the level for free beta (HCG) in: (1)pregnant women carrying Down syndrome fetuses and (2) pregnant womencarrying normal fetuses, said comparison being indicative of saidpregnant woman's risk of carrying a fetus with Down syndrome.